Competitive binding immunoassays for measuring ligands are well known, and are based on the competition between a ligand in a test sample and a labeled reagent, referred to as a tracer, for a limited number of receptor binding sites on antibodies specific to the ligand and tracer. The concentration of ligand in the sample determines the amount of tracer that will specifically bind to an antibody. The amount of tracer-antibody complex produced can be quantitatively measured and is inversely proportional to the quantity of ligand in the test sample.
Fluorescence polarization immunoassay techniques are based on the principle that a fluorescent labeled compound when excited by plane polarized light, will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Specifically, when a molecule such as a tracer-antibody complex having a fluorescent label is excited with plane polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time when light is absorbed and when it is emitted. When a "free" tracer compound (i.e., unbound to an antibody) is excited by plane polarized light, its rotation is much faster than that of the corresponding tracer-antibody complex; therefore, the emitted light is depolarized to a much greater extent. Thus, the molecular rotational relaxation time, and hence the magnitude of the fluorescence polarization response, is directly related to the molecular size of the compound. Accordingly, when plane polarized light is passed through a solution containing a relatively high molecular weight fluorescent compound, the degree of polarization of the emitted light will in general be greater than when plane polarized light is passed through a solution containing a low molecular weight fluorescent compound. Thus, fluorescence polarization provides a quantitative means for measuring the amount of tracer-antibody complex produced in a competitive binding immunoassay.
The fluorescence polarization principle is ordinarily utilized in an assay by mixing a sample containing an analyte or ligand of interest (or suspected of containing an analyte) with a "tracer", i.e., a labelled compound similar to the analyte but capable of producing a fluorescence polarization response to plane polarized light. Conventionally, the analyte or ligand is a relatively low molecular weight compound, i.e. less than about 2,000 daltons, but the analyte may be substantially larger, e.g., having a molicular weight on the order of 100,000 daltons or more, as long as it is capable of measurement using fluorescent polarization immunoassay techniques (see, for example, co-pending U.S. application Ser. No. 757,822 filed July 22, 1985, the disclosure of which is incorporated herein by reference, which describes a fluorescent polarization immunoassay for C-reactive protein having a molecular weight of about 120,000 daltons). Antibody specific to the analyte and the tracer is also included in the mixture. The tracer and the ligand compete for a limited number of receptor binding sites on the antibody. The amount of tracer that will bind is inversely related to the concentration of analyte in the sample, since the analyte and tracer each bind to the antibody in proportion to their respective concentrations.
The TDx@ Fluorescence Polarization Analyzer, an instrument commercially available from Abbott Laboratories, Abbott Park, Ill., is an automated system for the performance, inter alia, of fluorescence polarization assays. The TDx@ Analyzer has achieved remarkable commercial success in providing fluorescent polarization immunoassays to clinical laboratories for the determination in patient samples of many ligands, including antiasthmatic drugs, such as theophylline, antiarrhythmic drugs, such as lidocaine, N-acetylprocainamide, procainamide and quinidine, antibiotic drugs, such as amikacin, gentamicin, kanamycin, netilmicin, streptomycin, tobramycin and vancomycin, anticonvulsant drugs, such as carbamazepine, phenytoin, phenobarbital, primidone, and valproic acid, antineoplastic drugs, such as methotrexate, cardiac glycosides, such as digoxin, thyroid function assays, such as T-uptake and thyroxine, and others. The TDx@ Analyzer and its use in the performance of immunoassays is described in Jolley, et al., "Fluorescence Polarization Immunoassay I. Monitoring Aminoglycoside Antibiotics in Serum and Plasma", Clinical Chemistry 27/7. 1190-1197 (1981); Popelka, et al., "Fluorescence Polarization Immunoassay II. Analyzer for Rapid, Precise Measurement of Fluorescence Polarization with Use of Disposable Cuvettes", Clinical Chemistry 27/7, 1198-1201 (1981); Jolley, et al., "Fluorescence Polarization Immunoassay III. An Automated System for Therapeutic Drug Determination", Clinical Chemistry 27/9, 1575-1579 (1981); and Lu-Steffes, et al., "Fluorescence Polarization Immunoassay IV. Determination of Phenytoin and Phenobarbital in Human Serum and plasma", Clinical Chemistry 28/11, 2278-2282 (1982).
One problem encountered in the use of fluorescent polarization immunoassay techniques to determine analytes of interest in serum or plasma samples is background fluorescence present in varying degrees in the samples. Icteric serum or plasma can contribute a significant error to the desired polarization measurement. A major fluorescent component of icteric serum or plasma is albumin-bound bilirubin. Bilirubin is the final product of heme catabolism and in normal individuals is present in serum at less than 1 mg/dl. In various disease states affecting the liver, bilirubin is markedly elevated, reaching 10-20 mg/dl in some cases. Neonates ofter attain high levels in the 10-20 mg/dl range due to poor liver function immediately post-partum. Bilirubin is relatively nonfluorescent when it is in aqueous solution, but becomes highly fluorescent if bound to albumin (Chen, Arch. Biochem. Biophys. 160, 106-112) and bilirubin-albumin binding is very tight (Gray, et al., J. Biol. Chem. 253, 4370-4377). Therefore, serum or plasma samples with elevated bilirubin levels will exhibit an elevated fluorescence due to the presence of the bilirubin-albumin complex.
In order to avoid erroneous results due to background fluorescence, a blank reading is usually taken on a serum or plasma sample in the TDx Analyzer prior to performance of an assay, which is then subtracted from the final assay reading to arrive at a corrected value. However, background subtraction may be ineffective to adequately compensate for background fluorescence in some elevated bilirubin samples, and degredation of the bilirubin-albumin complex during the course of the assay can result in inaccurate compensation for background fluorescence in such samples. Accordingly, U.S. Pat. No. 4,492,762 discloses conducting fluorescent polarization immunoassays in a solution containing effective amounts of an anionic surfactant to disrupt bilirubin-serum albumin complex in the sample and thereby reduce background fluorescence of the sample. The '762 patent discloses that a broad category of anionic surfactants are useful for this purpose and that concentration ranges of 0.001 to 0.2 (weight/volume) percent are preferred. Preferred surfactants for use in the practice of the method of the '762 patent have been sodium dodecyl sulfate and sodium cholate. Although the method of the '762 patent has proven to be highly effective in limiting measurement errors on the TDx. Analyzer due to elevated bilirubin levels in serum samples, the use of such surfactants has precluded the development of fluorescence assays for some ligands, such as where sensitivity requirements and large sample volumes are needed due to low concentrations of the ligand in the sample being tested. In these cases, the use of sodium dodecyl sulfate or other surfactants disclosed in the '762 patent would require sufficiently high levels of surfactant as to result in antibody or analyte degredation in the performance of the assay. In order to overcome this problem for low concentrations of relatively low molecular weight ligands, such as therapeutic drugs like digoxin, a harsh pretreament step has been required to remove bilirubin interference in fluorescence polarization immunoassays, for example by denaturing proteins in the serum. For low concentrations of relatively large molecules, such as proteins, such pretreatments can not be used since they would result in denaturation of the analyte in the sample, and fluorescence polarization immunoassays for such relatively large molecules using fluorescence polarization techniques has not been possible. These and other problems with prior art systems are overcome by the practice of the present invention wherein fluoresence polarization immunoassays for analytes in serum or plasma samples are conducted in the presence of dioctyl sodium sulfosuccinate.